Valproic Acid Improves Glucose Homeostasis by Increasing...

J BIOCHEM MOLECULAR TOXICOLOGYVolume 30, Number 9, 2016

Valproic Acid Improves Glucose Homeostasis byIncreasing Beta-Cell Proliferation, Function, andReducing its Apoptosis through HDAC Inhibitionin Juvenile Diabetic RatSabbir Khan and Gopabandhu JenaFacility for Risk Assessment and Intervention Studies, Department of Pharmacology and Toxicology, National Institute of PharmaceuticalEducation and Research, S.A.S. Nagar, Punjab 160062, India; E-mail: [email protected], [email protected]

Received 27 January 2016; revised 1 March 2016; accepted 11 March 2016

ABSTRACT: Recent evidence highlighted that thereis a link between type-1 diabetes mellitus and his-tone deacetylases (HDACs) due to their involvementin beta-cell differentiation, proliferation, and function.The present study aimed to investigate the protectiverole of valproic acid (VPA) on beta-cell proliferation,function, and apoptosis in juvenile diabetic rat. Dia-betes was induced in juvenile Sprague–Dawley rats bystreptozotocin (75 mg/kg, i.p.) and VPA was adminis-tered at the doses of 150 and 300 mg/kg/day for 3 weeksby oral route. Various biochemical parameters, cellularalterations, and protein expression as well as apopto-sis were assessed using different assays. VPA treatmentsignificantly decreased plasma glucose, beta-cell dam-age, and apoptosis as well as increased the beta-cellfunction, insulin level/expression. The present studydemonstrated that VPA improves beta-cell prolifera-tion and function as well as reduces beta-cell apoptosisthrough HDAC inhibition. Our findings provide evi-dence that VPA may be useful for the treatment of juve-nile diabetes. C© 2016 Wiley Periodicals, Inc. J. Biochem.Mol. Toxicol. 30:438–446, 2016; View this article online atwileyonlinelibrary.com. DOI 10.1002/jbt.21807

KEYWORDS: Apoptosis; Beta-Cell; Diabetes; HDACinhibitor; Sodium Valproate

Correspondence to: Gopabandhu JenaSupporting Information is available in the online issue at

www.wileyonlinelibrary.com.Contract grant sponsor: National Institute of Pharmaceutical

Education and Research.C© 2016 Wiley Periodicals, Inc.

INTRODUCTION

Type-1 diabetes mellitus (T1DM) is a chronic au-toimmune disorder characterized by hyperglycemiadue to compromised insulin secretion from beta-celland/or reduced beta-cell mass, which generally de-veloped in genetically susceptible individuals by en-vironmental factors [1, 2]. T1DM usually occurs inyounger people and termed as juvenile-onset diabetes,even if it can occur at any age [3]. According to In-ternational Diabetes Federation (IDF), T1DM has lowprevalence (5%–10%), however its incidence increasesgradually (2%–3%/year), particularly in younger (<15years) people [4]. Both genetic and epigenetic factorscontributed equally in the pathogenesis of T1DM [5, 6].Epigenetic alterations such as post-translational modi-fication, DNA methylation and non-coding RNAs playa critical role in the development of pancreas, cellulardifferentiation, proliferation, and functions [7].

Recent evidence suggested that there is a linkbetween T1DM and histone deacetylases (HDACs)because HDAC inhibitors contribute in the beta-cellproliferation and function [8–10]. Over expression ofHDACs can modulate the lineage control of pancre-atic islet cells [10, 11]. Further, HDAC inhibitors en-hance and maintain the expression profile of the pro-endocrine markers in the pancreas [11, 12]. Moreover,regulation of insulin gene transcription is also modu-lated by acetylation status of histone, suggesting the es-sential role of HDACs in insulin synthesis/expression[9]. Both clinical and experimental studies have beenshown that apoptosis is the primary cause of beta-celldeath in T1DM through various signaling [3]. HDACinhibitors protect beta-cell from various pathologicalinsults such as cytokine-induced damage and apop-tosis in vitro and in vivo [8, 12, 13]. HDAC inhibitors

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Volume 30, Number 9, 2016 VALPROIC ACID IMPROVES BETA-CELL FUNCTION 439

also suppress virus-induced inflammatory responsesand progression of T1DM in rats [14]. Recently, wehave reported that HDAC inhibition by sodium bu-tyrate protects beta-cell damage and apoptosis as wellas improve glucose homeostasis in juvenile diabeticrats [15].

Valproic acid (VPA) is a first-line drug used for thetreatment of seizures, migraine, and bipolar disorders.VPA has been proven as a HDAC inhibitor and prefer-ably subdued the activities of class I and II HDACs[16, 17]. It is a matter of debate that VPA modulatesinsulin synthesis/secretion, body weight, and insulinsignaling, but the exact mechanism remains unknown[18]. It has been reported that VPA directly enhancesthe insulin secretion in epilepsy patients [19]. Recently,Manaka et al. [20] have been reported that chronicexposure of VPA promotes insulin release by reduc-ing KATP channel current without affecting Ca2+ sig-naling in mouse islets. Further, acute VPA treatmentalso reduces blood glucose by potentiating insulin ac-tion in streptozotocin (STZ)-induced T1DM in mice[21]. It has also been reported that VPA exerts hypo-glycemic, anti-lipidemic, and anti-oxidant in alloxan-induced type-1 diabetic rat [22]. Interestingly, a clini-cal study also showed glucose-lowering effect of VPAby single i.v. administration during oral glucose tol-erance test in patients with newly diagnosed epilepsy[23]. Recently, we have reported the renoprotective andanti-fibrotic effects of VPA in T1DM through HDACinhibition [24, 25]. Additionally, VPA also reduced theinsulin-resistance, fat accumulation, dyslipidemia, andhepatic-glucose production in type-2 diabetic rat, sug-gesting its effects on peripheral tissues like liver andadipose tissue [26]. Therefore, here we hypothesizedthat VPA can modulate the beta-cell mass, insulinsynthesis/release, and might improve glucose home-ostasis in T1DM through HDAC inhibition and/orassociated mechanisms in STZ-induced T1DM in ju-venile rat. The present study was planned in juve-nile animals to mimic the possible clinical pathogen-esis of T1DM, as it generally occurred in younger agepeople.

MATERIALS AND METHODS

Animals

Animals were procured from the Central AnimalFacility of the institute and experimental protocol wasapproved by the Institutional Animal Ethics Commit-tee (IAEC). All the experiments were performed onjuvenile Sprague–Dawley rats in accordance with theCommittee for the Purpose of Control and Supervisionof Experimentation on Animals (CPCSEA) guidelines.

Animals were kept under controlled environment atroom temperature (22 ± 2°C) and humidity (50 ± 10%)with automatically controlled 12 h light and dark cycle.Feed and water were provided ad libitum. Animals wereacclimatized at least for 3 days prior to commencementof the experiment.

Chemicals and Reagents

Sodium valproate (CAS No. 1069-66-5, pu-rity >98%), D-glucose, sucrose, tris(hydroxymethyle)aminomethane (tris) base, trichloroacetic acid (TCA),SDS, proteases inhibitor cocktail, and dibutyl phthalatein xylene were purchased from Sigma–Aldrich chem-icals (Saint Louis, MO). STZ, EDTA, sodium fluoride,triton-X-100, and glass slides were purchased from Hi-media (Mumbai, India). Primary and secondary an-tibodies, BSA, and luminol reagent were purchasedfrom Santa Cruz Biotechnology (Dallas, TX, USA),while other routine laboratory reagents such as ethanol,NaCl, xylene, and formalin were purchased from localsuppliers.

Experimental Design and Animal Treatment

All the animals (90–120 g) were randomized intofive groups consisting of 10 animals in each group.Group-1, control receiving saline; group-2, VPA con-trol (VPA300), receiving VPA 300 mg/kg/day for3 weeks by oral route; group-3, diabetic (D) induced byi.p. injection of STZ (75 mg/kg); group-4, (D+VPA150),diabetic rats treated with VPA at the dose of 150 for 3weeks by oral route; and group-5, (D+VPA300), dia-betic rats treated with VPA at dose of 300 mg/kg/dayfor 3 weeks by oral route. All animal were sacrifice af-ter 24 h of the administration of last dose. The doses ofVPA 150 and 300 mg/kg were selected on the basis ofprevious studies [24, 27–29].

Quantification of Biochemical Parameters

The plasma glucose,serum glutamic oxaloacetic(SGOT) or aspartate transaminase (AST) and serumglutamate-pyruvate transaminase (SGPT), or alaninetransaminase (ALT) were quantified by commerciallyavailable kits (ACCUREX, Mumbai, India), whileplasma insulin was quantified by ELISA kit (Mercer Ex-pert Assays, Los Angeles, CA, USA) according to man-ufacturer’s instructions. The HbA1c was quantified bycommercially available kit (Coral Clinical System, Goa,India) according to manufacturer’s instructions and re-sults were expressed as % HbA1c.

J Biochem Molecular Toxicology DOI 10.1002/jbt

440 KHAN AND JENA Volume 30, Number 9, 2016

TABLE 1. Effect of Diabetes and VPA Treatment on the Body and Organ Weights, Plasma Glucose, and Insulin Levels as wellas HbA1c in Juvenile Rat after 21 Days Treatment

Groups

Parameters Con VPA300 Diabetes D+VPA150 D+VPA300

Change in body weight (g) 82.90 ± 6.37 85.90 ± 8.10 −19.29 ± 6.49***ab −3.57 ± 4.83 5.33 ± 7.72Pancreas weight (g) 0.62 ± 0.03 0.61 ± 0.04 0.41 ± 0.01***ab 0.47 ± 0.02 0.53 ± 0.05Liver weight (g) 9.00 ± 0.35 9.04 ± 0.44 6.00 ± 0.26***ab 6.48 ± 0.18 6.49 ± 0.41Plasma glucose (mg/dL) 116.06 ± 4.06 110.67 ± 3.80 620.02 ± 20.54***ab 610.45 ± 4.02 455.67 ± 69.99**c

Plasma insulin (ng/mL) 2.55 ± 0.16 2.42 ± 0.29 0.45 ± 0.11***ab 0.96 ± 0.23 2.25 ± 0.21***c

%HbA1c 4.48 ± 0.25 4.32 ± 0.15 7.92 ± 0.70***ab 7.60 ± 0.44 6.88 ± 0.23

All the values are expressed as mean ± SEM (n = 5–10), ***p < 0.001 and **p < 0.01, “a” versus control “b” versus VPA control, and “c” versus diabetic control.“−“show decreased in body weight (difference between final and initial body weight).

Glucose Tolerance Tests

Intraperitoneal glucose tolerance test was per-formed as described by Khan and Jena with some mod-ifications [15]. In brief, animals were fasted for 6–8 hthen D-glucose (2 g/kg) was administered by i.p. injec-tion and blood samples were collected at 0, 15, 30, 60,and 120 min and plasma glucose were measured. To-tal AUC were calculated using the trapezoidal method[30] and expressed as % of control.

Histology and Immunohistochemistry

The pancreas and liver were fixed in 10% neutralbuffer formalin and histological slides were preparedaccording to standardized protocol in our laboratoryand described previously [31] with some modificationsusing commercially available kit NovolinkTM PolymerDetection System (Leica, Milton Keynes, UK) accord-ing to manufacturer instruction’s using the primaryantibodies against the insulin, proliferating cell nuclearantigen (PCNA), and acetylated histone H3 (Santa CruzBiotechnology). Slides were examined using Olym-pus microscope (Model BX 51; Tokyo, Japan). Ran-domly 15–20 fields or focuses from each animal wereobserved and quantified by Image J (version 1.46m)software.

Evaluation of Apoptosis in Islets by TUNELAssay

Pancreas was fixed in the chilled 10% formalin,embedded in paraffin, and cut into 5 μm thick sec-tions. Terminal deoxynucleotidyl transferase-mediateddUTP nick end labeling (TUNEL) assay was used toassess the DNA fragmentation (Catalogue No. QIA39;Calbiochem, Oncogene Research Product, La Jolla, CA,USA). The assay was performed according to the man-ufacturer’s instructions and total cell population and

TUNEL positive cells were counted using the imageanalysis software ‘Isis’ (Carl Zeis, Axioimager M1,Germany) and images were acquired using chargedcoupled device camera and expressed as % apoptoticcells.

Histone Extraction and Immunoblotting

The histone/nuclear protein extraction wasperformed as previously described [15] with somemodification. Approximately one (g) of tissue washomogenized in 5 mL of low sucrose (12%) buffer(buffer-A) containing protease inhibitors and ho-mogenate was carefully layered on 5 mL high sucrose(15%) buffer (buffer-B) containing inhibitors andcentrifuged at 660 × g for 10 min. Cellular pellet wassuspended in buffer-A with a detergent and layeredon buffer-B then centrifuged at 660 × g for 10 minresulted nuclear pellet was again suspended, layered,and centrifuged. Final pellet was dissolved in low saltbuffer (LSB) with NP-40 and sonicated three times for10 s then centrifuged at 18000 × g for 10 min. The pelletwas dissolved in LSB containing 0.25 M hydrochloricacid with sonication and kept for 30 min in ice then cen-trifuged at 27000 × g for 30 min. The supernatant wasprecipitated with TCA and centrifuged at 18,000 rpmfor 30 min. The pellet was washed with acetone con-taining HCl and subsequently with pure acetone, thenhistones was dissolve in water and sample for SDS-PAGE was prepared. Equal amounts of proteins wereloaded in 14% SDS-PAGE and transferred onto nitro-cellulose membrane. Immunoblotting was performedusing anti-acetylated histone H3 and total histoneH3 primary antibodies with horseradish peroxidase-conjugated secondary antibodies. Protein signalswere detected by enhanced chemiluminescence andquantified with the help of Imagequant TL software(Imagequant 350; GE Healthcare, Hong Kong, China).



FIGURE 1. Effect of diabetes and VPA treatment on (A) survival rate, (B and D) glucose tolerance including area under curve (AUC) after achallenge dose of D-glucose. (C) Representative photomicrographs of histological alterations in islets stained with hematoxylin and eosin (H&E),magnification 1000×. Dashes lines show severely damage islets in diabetic pancreas. All the values are expressed as mean ± SEM, (n = 6–10).***p < 0.001 and *p < 0.05, “a” versus control, “b” versus VPA control and “c” versus diabetic control.

Statistical Analyses

Results are shown as mean ± standard error ofmean (SEM) for each group. Statistical analysis wasperformed using SigmaStat (Version 3.5) statistical soft-ware (Systat Software, San Jose, CA, USA). One wayANOVA was used to determine the level of signifi-cance among the difference groups, and post-hoc anal-ysis was performed using Tukey’s test, and p < 0.05considered to be statistically significant.

RESULTS

Effect of VPA on Body and Organ Weight aswell as Mortality/Survival

The progression of diabetes significantly de-creased the body weight (weight gain) gradually with

time as compared with the non-diabetic animals, whileVPA treatment restored the weight gain, but it wasstatistically insignificant (Table 1 and Supp. FigureS1). Similarly, VPA treatment restored the diabetes-induced decreased liver and pancreas weight as com-pared with respective controls, but it was statisticallyinsignificant (Table 1). Moreover, VPA treatment de-creased/delayed the diabetes-associated mortality rateas compared with respective controls (Figure 1A).

Effect of VPA on Glucose, HbA1c, andGlucose Tolerance

VPA treatment significantly decreased the diabetes-induced plasma glucose as compared to controls(Table 1). VPA treatment failed to decrease diabetes-associated increase in the HbA1c level as comparedwith respective controls (Table 1). Diabetes led to



FIGURE 2. Effect of diabetes and VPA treatment on the beta-cell proliferation and insulin expression in pancreas of juvenile rat. Representativephotomicrographs of IHC showing expression of insulin and PCNA in islets, magnification 1000×. Arrows show the PCNA positive cells.Dashes-line circles show islets. All the values are expressed as mean ± SEM (n = 5). ***p < 0.001 and **p < 0.01, “a” versus control, “b” versusVPA control and “c” versus diabetic control.

significantly impair the glucose tolerance and clear-ance following single challenge dose of glucose, whileVPA (300 mg/kg) significantly improved the impairedglucose tolerance as compared with respective controls(Figures 1B and 1D).

Effect of VPA on Histological Alterations inIslets (Endocrine Pancreas)

Diabetes induced significant histological alter-ations particularly in the endocrine pancreas as com-pared with normal control. Approximately 70%–80%obliteration of endocrine cells (beta-cell) in islets as well

as decreased size of islets was observed in diabetic ani-mals as compared with control (Figure 1C). VPA treat-ment significantly ameliorated the above histologicalalterations such as decreased islets size and beta-celldamage as compared with diabetic control (Figure 1Cand Supp. Figure S2).

Effect of VPA on Beta-Cell Proliferation,Function, and Apoptosis

To evaluate the beta-cell proliferation and func-tion the expression of PCNA and insulin were evalu-ated in the pancreas by immunohistochemistry (IHC).



FIGURE 3. Effect of diabetes and VPA treatment on the beta-cell apoptosis in islets of juvenile rat. Representative photomicrographs of IHCshowing TUNEL positive cells (apoptostic) in islets, magnification 1000×. Arrows show the TUNEL positive (apoptotic) cells. Dashes-line circlesshow islets. All the values are expressed as mean ± SEM (n = 5). ***p < 0.001, “a” versus control, “b” versus VPA control and “c” versus diabeticcontrol.

Results revealed that diabetes significantly decreasedthe expression of both PCNA and insulin, whileVPA treatment significantly increased the same in adose-dependent manner as compared with respec-tive controls (Figure 2). Further, VPA treatment alsosignificantly reduced the diabetes-associated beta-cell apoptosis as evident by TUNEL assay results(Figure 3). The above results confirmed that VPA treat-ment restored diabetes-induced decreased plasma in-sulin level as well as histological alteration in isletsas compared with respective controls (Table 1 andFigure 1C).

Effect of VPA on HDAC Inhibitionand Histone Acetylation

To evaluate the HDAC inhibition potential of VPAin the present experiment acetylation of histone H3was evaluated by Western blotting and IHC in pan-creas. IHC results also confirmed that VPA treatmentsignificantly restored the acetylation of histone H3in diabetic rat as compared with respective controls(Figure 4A). Further, VPA treatment significantlyincreased the diabetes-associated decreased acetyla-tion of histone H3 in the pancreas as compared withrespective controls (Figures 4B and 4C). Moreover,VPA per se increased the acetylation of histone H3

in non-diabetic animals as compared with untreatedanimals (Figures 4B and 4C).

DISCUSSION

The present study demonstrated that VPA treat-ment significantly decreased the plasma glucose,HbA1c, beta-cell apoptosis, and improved glucoseclearance and insulin synthesis/expression as wellas delayed mortality rate, which confirmed its anti-diabetic role. The histological evaluation also revealedthat VPA treatment significantly ameliorated thebeta-cell damage. In the pancreas, HDACs is tightlycontrolled at normal physiology and modulates itsdevelopment and cellular differentiation, therebyfunction [11]. HDAC inhibitors can increase beta-cellmass through modifying the differentiation andproliferation as well as reducing its apoptosis againstinflammatory cytokines in STZ-induced diabetesin rodents and INS-1 cells [11, 13, 15]. IHC resultsindicated that VPA significantly increased the beta-cellproliferation and function as revealed PCNA andinsulin expression. Thus, our results indicated thatVPA exerts protective effect in T1DM by improvingbeta-cell mass/function at biochemical and structurallevel. The possible mechanism for the protective effectsof VPA might be HDAC inhibition and subsequentmodulation of various genes and transcription factors



FIGURE 4. (A) Representative photomicrographs of IHC showing the effect of diabetes and VPA treatment on the expression of acetylation ofhistone H3 in islets, magnification 1000×. Arrows show the acetylated histone H3 positive cells, while dashes-line circles show islets. (A and B)Representative western blots showing the effect of diabetes and VPA treatment on the expression of acetylation of histone H3 in pancreas. Allthe values are expressed as mean ± SEM (n = 5). ***p < 0.001, **p < 0.01 and *p < 0.05, “a” versus control, “b” versus VPA control and “c} versusdiabetic control.

associated with T1DM. These findings supported byprevious report that butyrate reduces the beta-cellapoptosis and improves glucose homeostasis by mod-ulating p38/ERK signaling through HDAC inhibitionin rat [15]. Although, VPA is a first-line drug for thetreatment of epilepsy, but it has adverse effects suchas GI disturbances, hepatotoxicity, and reproductivetoxicity generally observed during chronic therapy[28, 29]. The present results also confirmed that VPAtreatment did not induce any hepatotoxicity as re-vealed by ALT, AST levels, and histological observation(Supp. Figure S3).

HDAC inhibitors attenuated the expressionof pro-apoptotic proteins and beta-cell damage bypreventing interleukin-1β (IL-1β)-induced activationof NF-κB and apoptosis signaling in experimentalstudies [12, 13]. In the present study, VPA treatment

significantly reduced the diabetes-associated beta-cell apoptosis, thereby increased its number/mass,which also supported by PCNA expression. The nextobvious question is whether the increased beta-cellnumber/mass is functional or not, insulin expressionwas evaluated by IHC. Our findings indicated thatVPA treatment significantly increased the insulinexpression and plasma insulin level, which assuredthat the increased beta-cells are fully functional (Figure2 and Table 1). These findings are in agreement with theprevious report, which highlighted that VPA directlyenhances the insulin secretion in epileptic patients[19]. HDACs play a critical role in the regulation ofsynthesis/expression of insulin by modulating thehistone acetylation [9, 32]. Thus, HDAC inhibition byVPA might contribute toward increase insulin level.It is worthy to mention that chronic exposure of VPA



facilitates insulin release by reducing KATP channelcurrent in mouse islets [20]. Moreover, VPA adminis-tration decreases the glucose level in spontaneouslydiabetic BB/E rats by modulating carbohydrate and fatmetabolism [33]. Further, VPA ameliorated the diabeticnephropathy, renal fibrosis, and pancreatic damage byinhibiting iNOS/NF-κB signaling as well as facilitat-ing autophagy through HDAC inhibition [24, 25, 34].Together, HDAC inhibition by VPA might be one ofthe most possible mechanisms for increased beta-cellproliferation and function as well as improved glucosehomeostasis. Additionally, VPA is a pleiotropic agentand acts on multiple targets, which might be partlycontributed to its protective role in diabetes [35].

The present study demonstrated that VPA in-creased the beta-cell proliferation, function, and re-duced the beta-cell apoptosis. Further, VPA treatmentalso reduced the plasma glucose, HbA1c, and increasedinsulin production/expression through HDAC inhibi-tion and might be beneficial for the treatment of juve-nile diabetes.

SUPPORTING INFORMATION

Figure S1. Effect of diabetes and VPA treatment onbody weight of juvenile rats during 3 weeks treatment.All the values are expressed as mean±SEM, (n=6-10).***p<0.001, ‘a’ vs. control and ‘b’ vs. VPA control.

Figure S2. Effect of diabetes and VPA treatment onhistological alterations in pancreas after 3 weeks treat-ment. Representative photomicrographs of histologicalalterations in islets stained with hematoxylin and eosin(H&E), magnification 400x.

Figure S3. Effect of diabetes and VPA treatment onthe histological alterations and liver function marker(ALT and AST). (A) Representative photomicrographsof histological alterations in islets stained with hema-toxylin and eosin (H&E), magnification 400x. (B and C)Plasma levels of ALT and AST after 3 week treatmentof VPA. All the values are expressed as mean±SEM,(n=6).

CONFLICT OF INTEREST

The authors declare that there are no conflicts ofinterest.

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